Field of the Invention
The invention pertains to the fields of cancer diagnosis and therapy. It relates to methods and kits for detecting and quantifying the expression of alternatively spliced Lamin C and Lamin A mRNAs, and especially to determination of the ratio of Lamin C mRNA to Lamin A mRNA in a biological sample. The inventor has found that this ratio is useful for identifying or classifying a neoplasm, such as breast cancer, and for selecting a treatment or treatment regimen for a patient having the neoplasm. Additional aspects of the invention relate to selecting a therapeutic method for treating a subject based on identification of the ratio of Lamin C mRNA to Lamin A mRNA.
Description of Related Art
The description of the related art provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventor, to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, is neither expressly nor impliedly admitted as prior art against the present invention.
The Lamin A/C gene encodes several lamin nuclear proteins through alternative splicing of mRNA transcribed from this gene. Nuclear lamins, also known as class V intermediate filaments, are fibrous proteins that make up the nuclear lamina matrix. The lamina is about 10 nm thick and underlies the inner nuclear membrane, which is reversibly broken-down during mitosis by way of phosphorylation. The major function of the nuclear lamina is to maintain structure and integrity of the nucleus. Lamins are highly dynamic in nature and are implicated in the nonrandom positioning of sub chromosome domains. They contribute to the size, shape, and overall mechanical stability of the nucleus and are involved in the overall organization of chromatin, regulating the cell cycle, DNA replication, transcription, cell differentiation, apoptosis and aging; Broers, et al., J. Cell. Sci. 112(Pt 20): 3463-75 (1999); Taddei, et al., Annu. Rev. Genet. 38:305-45 (2004); Shimi, et al., Adv. Exp. Med. Biol. 773:415-30 (2014).
Alternative splicing of mRNA transcribed from the Lamin A/C gene (also known as the LMNA gene) produces several different Lamin mRNA splice variants that encode the Lamin A, Lamin C, Lamin AΔ10, Lamin AΔ50 (Progerin), and Lamin C2 proteins. Lamin C mRNA is a transcript variant missing all of exons 11 and 12 present in Lamin A mRNA. Lamin AΔ10 mRNA is an alternative splicing product of the Lamin A/C gene lacking exon 10 and has a relatively low abundance compared to Lamin A and C mRNA; Machiels, et al., J. Biol. Chem. 271: 9249-53 (1996). Progerin mRNA is missing 150 nucleotides from the end of exon 11 (and the corresponding protein has a deletion of 50 amino acids near the C-terminal) and Lamin C2 mRNA is specific to the testis; Cao, et al., J. Hum. Genet. 48:271-4 (2003); De Sandre-Giovanni, et al., Science 300:2055 (2003); Eriksson, et al., Nature 423:293-8 (2003).
Lamin A and Lamin C, which are encoded by two of the above splice-variants, are two fibrous nuclear proteins that are translated from alternative mRNA splice variants of the same Lamin A/C (LMNA) gene which contains 12 exons.
Lamin A was identified as a biomarker colonic cells by Willis et al. who also found that Lamin A was upregulated in colorectal cancer (CRC) cells. Upregulation of Lamin A was associated with increased invasiveness and motility of CRC cells, but not with cellular proliferation. This was suggested as creating a more aggressive stem cell-like phenotype correlating with a two fold increase in mortality; Willis, et al., PLOS One 3:e2988 (2008); Willis, et al., Biochem. Soc. Trans. 36:1350-3 (2008). On the other hand, the study by Willis et al. detected Lamin A in the stem cell niche but not in transit amplifying cells. Lamin C, encoded by a different splice variant, was not found in either the stem cell niche or in transit amplifying cells; Willis, et al., PLOS One 3:e2988 (2008). Controversially, low expression of Lamin A was associated with increased disease recurrence in stage II and III colon cancer patients Belt, et al., Eur. J. Canc. 47: 1837-45 (2011).
Past research has studied Lamin A and Lamin C collectively assuming that both proteins function as one. It was not known whether these proteins act in concert or have distinct roles and associations in cancer cells. Although Lamin A and Lamin C only differ by 98 residues, recent studies have illustrated the unique roles of Lamin A and Lamin C. Structurally, Lamin C is unique to the other Lamins in that it is the only Lamin lacking the carboxy-terminal sequence that is required for membrane attachment during its biogenesis and trafficking to the nucleus (Al-Saaidi, et al., Chromosoma 124(1):1-12 (2014) and is found exclusively in mammals; Peter, et al., Nucleus 3:44-59 (2002). It is hypothesized that this C-terminal end may play a role in senescence. Additionally, it was found that the tumor suppressor AIMP3/P18 to be involved in ubiquitination-dependent degradation of Lamin A, but not Lamin C, indicating that this C-terminal end may have various roles; Oh, et al., Aging Cell 9:810-22 (2010). Even though well studied, the difference still remains vague and poorly understood as apparent from the publications cited below.
Hung, et al., U.S. 2004/0018546 A1 proposes detecting various markers including those from nuclear matrix proteins such as Lamin A, Lamin B, and Lamin C in ductal fluid from breasts. Their focus is on use of ductal fluid and no correlations between expression of Lamin A, Lamin C or a ratio of Lamin C to Lamin A markers are shown.
Hutchison, et al., U.S. 2010/0297618 A1 describes a method for determining a prognosis of colorectal cancer by measuring Lamin A indicating that a loss or mislocalisation of A-type lamin proteins correlates with a positive prognosis while the presence of A-type lamin proteins is indicative of a poor prognosis.
Park, et al. U.S. 2011/0033873 A1 describes proteonomic markers for early detection of hepatocellular carcinoma. They propose that Lamin C (Lamin A/C transcript variant 2) can be used as a tumor marker.
Wazir, et al., Cell. Mol. Biol. Lett. 18(4): 595-611 (2013) studied associations among mRNA expression by Lamin A/C, Lamin B1 and Lamin B receptor. Wazir does not distinguish between different Lamin A/C transcript variants such as Lamin A (transcript variant 1) and Lamin C (transcript variant 2) which encode lamin proteins having different structures and functions.
The expression of Lamin A, Lamin C and other lamins was not believed to be a reliable biomarker due to differential expression of lamins in various tissues as shown in Table 1 and in view of conflicting or inconsistent experimental studies. Lamin C and Progerin have been shown to have distinct and opposite functions in relation to cellular energy expenditure and lifespan; Lopez-Mejia, et al., EMBO Reports 15:529-39 (2014). The variation in expression of Lamin A and Lamin C in different kinds of cells has been attributed to altered splicing, mRNA stability, translation efficiency, or protein stability; Al-Saaidi, et al., Chromosoma (2014)).
Lamins are expressed in well-differentiated cells and tissues among humans but not or poorly expressed in stem cells; Broers, et al., Histochem. Cell Biol. 107:505-17 (1997); Eckersley-Maslin, et al., Nucleus 4:53-60 (2013). Lamin A was found to be expressed highly in stem cells while Lamin C was found to be expressed in differentiated epithelial cells and smooth muscles of the colon; Willis, et al., Biochem. Soc. Trans. 36:1350-3 (2008). Previous reports indicated that the heart, kidney, and liver show similar levels of Lamin A and Lamin C, while skeletal muscles have higher expression of Lamin A and Lamin C than cardiac ones; Rober, et al., Development 105:365-78 (1989); Swift, et al., Science 341: 1240104 (2013). In contrast, a higher level of Lamin C compared to Lamin A has been reported in astrocytes, oligodendrocytes, and neurons and mouse retinal neurons, i.e., neuronal tissues; Jung, et al., Molec. Neurobiol. 47:290-301 (2013); Wakabayashi, et al., Histochem. Cell Biol. 136:427-36 (2011). The expression of Lamin A is reduced or absent in subsets of cells with a low degree of differentiation and/or cells that are highly proliferating including human malignancies; Broers, et al., Histochem. Cell Biol. 107:505-17 (1997); Rober, et al., Development 105:365-78 (1989); Hutchison, et al., Nat. Cell. Biol. 6:1062-7 (2004), especially leukemia and lymphomas; Stadelmann, et al., Leuk. Res. 14:815-21 (1990); Lin, et al., Exp. Cell Res. 236: 378-84 (1997).
It has also been illustrated that the loss of the Lamin A/C gene compromises the nuclear envelope and leads to breast cancer aberrations in nuclear morphology and aneuploidy; Capo-chichi, et al. Chinese J. Canc. 30:415-25 (2011). Epigenetic silencing of the Lamin A/C gene by CpG island promoter hypermethylation correlated with the loss of RNA and protein expression in leukemia and lymphoma malignancies; Agrelo, et al., J. Clin. Oncol. 23: 3940.7 (2005). Low expression of Lamin A was also found to be associated with increased disease recurrence in stage II and III in CRC patients; Belt, et al., Eur. J. Canc. 47:1837-45 (2011).
In view of these obstacles, and prompted by the need for early detection and accurate staging and monitoring of cancer, the inventor sought to develop a reliable biomarker based on the expression of lamins that was not limited to measuring the expression of a single type of lamin as a tumor or cancer biomarker and avoid the problems associated with variant expression of lamins in different tissues. Therefore, the coexpression of different lamin splice variants was studied to determine whether quantitative differences amongst lamin mRNA splice variants expression would provide reliable biomarkers for cancer or tumors. Surprisingly, the inventor found that the ratio of Lamin C mRNA to Lamin A mRNA to be a reliable biomarker of cancer for many different types of cancer. This provided a basis for new methods for early detection of cancer, for monitoring the effects of cancer chemotherapy and for significantly improving prognostic outcomes by increasing mean survival rates and times for patients.